DT-diaphorase (DTD) is an enzyme that is overexpressed in certain cancerous tissues, including breast, colon, liver, bladder, stomach, the central nervous system (CNS) and lung tumors and in melanomas. For example, the expression of DTD is increased up to 80-fold in primary non-small cell lung cancer (NSCLC) relative to normal lung and up to 400-fold in NSCLC relative to small cell lung cancer (SCLC) cell lines [Malkinson et al., Cancer Res., 52:4752-4757 (1992)]. Evidence to date suggests increased expression of DTD reflects increased activity of AP-1 binding proteins that positively regulate DTD transcription [Kepa et al., Cancer Res. 38:2515 (1997)].
Accordingly, it would be desirable to use a DTD-directed approach for cancer therapy. Ideally, the mechanism of action would involve the production of a reactive quinone pro-drug after being reduced by DTD. The reduced quinone pro-drug can then crosslink DNA, which leads to cell death.
Various quinone pro-drugs have been studied to determine their effect on cancer cells expressing DTD. For example, NSCLC xenografts with high DTD activity were more susceptible to the quinone pro-drug mitomycin C than SCLC xenografts with low DTD activity. Although mitomycin C has been shown to have activity in NSCLC, it has two major drawbacks as a useful therapeutic agent in a DTD-directed approach. First, mitomycin C is a very poor substrate for DTD [Beall et al., Cancer Res. 54:3196-3201 (1994)]. Second, metabolism of mitomycin C by DTD is pH-dependent leading to pH-dependent inhibition of DTD at higher pHs [Siegel et al., Mol. Pharmacol., 44:1128-1134 (1993)].
Another quinone pro-drug, E09, is efficiently reduced by DTD, but primarily generates reactive oxygen radicals rather than DNA crosslinks. E09 has been shown to exclusively form DNA strand breaks. Being a conventional "hypoxic-targeting" agent, E09 was not active in spheroid studies demonstrating increased expression of DTD towards the necrotic center of the spheroid. The results probably relate to the poor ability of E09 to penetrate cells and cross cellular membranes [Bibby et al., Int. J. Oncol., 3:661-666 (1993)]. Other conventional "hypoxic-targeting" agents include tirapazamine and the nitroimidazole compounds.
Diaziquone (AZQ) is also an extremely poor substrate for DTD and does not readily form DNA crosslinks after reduction by DTD as reported by Siegel et al., Cancer Res., 50:7293-7300 (1990). In fact, AZQ failed to show any selectivity for DTD-rich cell lines.
MeDZQ (2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone) was a promising agent that utilizes the DTD enzyme-directed approach to cancer treatment. MeDZQ: (i) is greater than 150 fold more effective as a substrate for recombinant human DTD than mitomycin C, and (ii) shows pH-independent metabolism and does not inactivate DTD at higher pHs [Beall et al., supra; Ross et al. Oncol. Res., 6:493-500 (1994)]. DNA crosslinking by MeDZQ showed an absolute requirement for reduction of the drug and sequence-selectivity studies demonstrated that the drug crosslinked DNA at GNC sequences [Lee et al., Biochemistry, 31:3019-3025 (1992)]. In addition, there was good correlation between the levels of DTD in NSCLC and breast cancer cell lines and the cytotoxicity of MeDZQ [Beall et al., Mol. Pharmacol. 48:499-504 (1995)]. In fact, recent data from the NCI tumor cell line panel/COMPARE analysis has shown that there is a good correlation between DTD activity and MeDZQ cytotoxicity across 69 human tumor cell lines (r=0.3033, p&lt;0.016).
Although MeDZQ undergoes bioreductive activation, it was designed to be bioactivated specifically by DTD. As such, it is distinctly different from E09, tirapazamine and the nitroimidazole compounds. Unlike these "hypoxic-targeting" compounds, MeDZQ only produces a very modest increase in cytotoxicity under hypoxic conditions relative to aerobic conditions.
CHO cell lines stably transfected with human DTD were produced as reported in Gustafson et al., Mol. Pharmacol., 50:728-735 (1996). The parent non-transfected cell lines had no detectable DTD activity, while the activity of the transfectants ranged from 19-3527 nmol/min/mg protein. The cytotoxicity of MeDZQ was markedly increased as a result of transfecting DTD into CHO cells. Studies in NSCLC xenografts confirmed the relationship between the levels of reducing enzymes and the efficacy of MeDZQ.
The formulation of MeDZQ as a useful therapeutic, however, was hampered by its poor solubility. Therefore, a need exists for anti-cancer agents that have the desirable properties of MeDZQ, but without its poor solubility. The present invention satisfies this need and provides related advantages as well.